Antigenic substances such as insulin, growth hormone and immunoglobulin are characterized by specificity and sensitivity with which they bind to their antibodies. Utilizing these features, numerous attempts have been made to measure such antigenic substances or their antibodies and in consequence a number of immunochemical measuring processes are now in practical use. For instance, there are; the immunodiffusion methods, in which the antigen and the antibody are reacted together in agar gel; agglulination reaction and agglutination inhibition reaction methods, in which blood cells or fine particles like polystyrene latex are employed as carrier of antigen or antibody; and radioimmunoassay (RIA), in which radioisotopes are employed to label the antigen or the antibody.
Meanwhile, low molecular substances such as steroids, thyroid hormones and active amines, the production of whose antibodies is difficult, used to be measured by the competitive method utilizing the binding reaction of the receptor protein or the binding protein which specifically binds to these substances. Recently, however, the antibodies of these substances have come to be produced with relative ease and accordingly they can now be measured in the same way as the above-mentioned antigenic substances.
These methods have respective characters, by virtue of which they find wide applications. Among others, RIA which is far superior in the sensitivity and quantitative precision of measurement is used widely, the substances measurable by it including a great variety of high molecular substances such as immunoglobulin, virus, protein hormone and low molecular ones such as peptides, steroids.
RIA utilizing radioisotopes, however, demands advanced knowledge and skill from the operator; calls for expensive instruments and facilities; is subjected to many restrictions on account of environment pollution; and for these reasons it fails to find wide applications in clinical practice. Meanwhile, when a substance to be labeled with a radioisotope is, for instance, a low molecular compound like a steroid or an unstable substance like the human placental lactogen, it is difficult to directly label the substance; and even if labeling is possible, the substance is found practically inapplicable due to a decrease or loss of the immunological activity of the substance.
To avoid these inconveniences, lately new processes have been developed such as EIA (enzyme immunoassay) utilizing an enzyme, and FIA (fluorescence immunoassay) utilizing a fluorescent material, instead of a radioisotope. These process are featured by the sensitivity and the quantitative precision of measurement which are approximately equivalent to those of RIA.
RIA, EIA and FIA work on the same principle, i.e., two methods; competitive method and sandwich method. The principle is to be described referring to the case of an antigen being employed as a substance to be measured and an antibody being employed as a substance which specifically binds to the substance to be measured.